Source: WASHINGTON STATE UNIVERSITY submitted to
BIOCHEMICAL AND MOLECULAR MECHANISMS OF RESISTANCE TO DECAY IN WEED SEEDS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1000669
Grant No.
2014-67013-21575
Project No.
WNP05277
Proposal No.
2013-02322
Multistate No.
(N/A)
Program Code
A1131
Project Start Date
Jan 1, 2014
Project End Date
Dec 31, 2018
Grant Year
2014
Project Director
Fuerst, E.
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
Crop and Soil Sciences
Non Technical Summary
Weedy and invasive species spread by means of seeds, and persist for years in the soil as dormant seeds. This work focuses on the dormant seeds, referred to as the 'weed seed bank'. The weed seed bank has proven to be an intractable barrier to long-term weed management. In this work we develop knowledge to help us understand how seeds resist decay organisms. Specifically we are determining the biochemical and molecular basis for seed defense responses to decay organisms. Methods: Biochemical defense responses will be determined by measuring induction of defense enzymes in dormant seeds by fungal pathogens; the potential mechanisms of induction will also be determined including measuring mRNA accumulation. These defense responses will be determined first in wild oat, our model weed, using a Fusarium avenaceum isolate that causes wild oat seed decay. Subsequent studies will utilize other fungi as well as seeds of other weed, crop, and native plant species. The potential for these fungi to induce defense responses and cause seed decay in wild oat will be determined in soil. Goal: The goal of this project is to increase our knowledge of interactions between seeds and soil microbes with the long-term goal of developing technology to use seed decay as an ecological process to manage weedy and invasive species.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21323001140100%
Knowledge Area
213 - Weeds Affecting Plants;

Subject Of Investigation
2300 - Weeds;

Field Of Science
1140 - Weed science;
Goals / Objectives
The goals of this project are to (1) elucidate biochemical and molecular defense responses to Fusarium strain 'F.a.1' in dormant wild oat caryopses, including (1A) characterize induction of the defense enzymes, (1B) determine the role of mRNA accumulation in defense enzyme induction, and (1C) determine the contribution of proteolytic activation of preexisting proteins; (2) evaluate seed decay and biochemical defense responses to diverse fungal pathogens in wild oat; (3) evaluate seed decay and biochemical defense responses in selected weed, crop, and native grass species; and (4) determine whether selected fungi cause wild oat seed decay in soil, with the long-term goal of developing organisms that promote selective weed seed decay in the field.
Project Methods
General Scientific methods: Biochemical defense responses will be determined by measuring induction of defense enzymes in dormant seeds by fungal pathogens; the potential mechanisms of induction will also be determined by measuring proteolytic activation and measuring mRNA accumulation using real-time PCR. The above responses will be first be determined in wild oat using our Fusarium avenaceum isolate that causes wild oat seed decay; subsequent studies will utilize other pathogenic fungi as well as seeds of other weed, crop, and native plant species. The potential for these fungi to induce defense responses and cause seed decay in wild oat will be determined in soil. Education and Outreach Efforts Graduate and Undergraduate Education: All students will be exposed to a great diversity of disciplines, working with plants, soil, and microorganisms, and will benefit from working in three laboratories having a wide range of expertise; they will present their work at laboratory meetings. Graduate students will present work at national meetings and publish their research. K-12 Education and Public Outreach: Dr. Kennedy presently leads an outreach program with hands-on science that reaches more than 600 students annually, encouraging students to think about science as something exciting in their lives and not just in books. We also have a history of working with regional organizations that reach the larger public, including native plant societies and state weed organizations. High School Interns: Drs. Fuerst and Okubara plan to have one intern per year in 2015 and 2016. The goals of the summer internship program are to stimulate interest in science, technology, engineering and mathematics (STEM) among pre-college Native American and Hispanic students from eastern and central Washington, and to provide them with a real-world experience in science. The program began in 2005 and currently accepts about 9 interns each summer. Most of these students have gone on to pursue college degrees and some have entered graduate school. Evaluation: Graduate students: The progress and success will evaluated in terms of publications, presentations, and ultimately by degrees awarded. Undergraduate students: The progress and success will be evaluated in terms of number of students employed and hours employed. K-12 Education: The progress and success will be evaluated in terms of the number of classrooms engaged and the number of students taught. Public Outreach: The progress and success will be evaluated in terms of the number of presentations and the number of people attending. High School Interns: the progress and success will be evaluated in terms of the successful completion of the internships through our program and ultimately by the placement of these interns in college programs and employment.

Progress 01/01/14 to 12/31/18

Outputs
Target Audience:The target audiences of this research include (1) weed scientists seeking to find ecological methods to manage weeds in food production systems, including managing the weed seed bank and (2) ecological researchers seeking to understand the role of microbes in seed survival and the possible alteration of microbial communities to improve establishment of desired plant species and/or reduce establishment of undesired species. Changes/Problems:There was amajor effort regarding Objective 1C(determine the contribution of proteolytic activation of preexisting proteins) for which we have no results to report at this time. The WSU core proteomics facility was given 36samples between March and July of 2018. The samples were run but results were totally inadequate. The facility has, for all practical purposes, been non-functional during 2018, through the current date (3-27-19). We still hope these samples will be analyzed and, given satisfactory results, we will write up, publish, and acknowledge NIFA support. There was also as major effort regarding Objective1B(determine the role of mRNA accumulation in defense enzyme induction), using a transcriptomic approach. Extraction of RNA was delegated to a collaborator and unfortunately all RNA was degraded and unacceptable for analysis. RNA extraction protocols are being optimized to limit degradation and RT-PCR experiments are planned for 2019 to finish this work.? What opportunities for training and professional development has the project provided?Dr. Lewis participated in theWorld Congress of Soil Science inRio de Janeiro, Brazil, South America. Dr. Okubara participated inPlant Animal Genome XXV, January 2018, San Diego, CA. How have the results been disseminated to communities of interest?Dr. Lewis presented results oftheWhole genome sequencing of Fusarium avenaceum strain F.a.1 at theWorld Congress of Soil Science. Rio de Janeiro, Brazil, South America. Dr. Okubara was principal author of a project report for the 2018 Dryland Field Day Abstracts for Washington State University and USDA-ARS research, Pullman, WA. Dr. Pollard presented results at two departmental seminars. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? (1) E.P. Fuerst and P.A. Okubara supervised Soil Science PhD student Anne Pollard in 2018. Anne completed her research, published two refereed papers, and defended her dissertation in December, 2018. Two manuscripts are in draft form, to be submitted in 2019. (2) T Sullivan supervised Crop & Soil Science postdoctoral research associate, Ricky W.Lewis, in 2018. Results were presented at an international meeting, and a manuscript is in draft form, to be submitted in 2019.Investigation was conducted concerning the fungal transcriptome responses to aluminum toxicity and the interaction between aluminum and F.a.1 and wild oat caryopsis colonization. Aluminum toxicity is particularly relevant to agriculture in eastern Washington, where in some regions soils are becoming increasingly acidified. (3) Goal 1A: Completed and published. Methods extended to soil assays in goal 4, below. (4) Goal 1B: Additional primers were developed to detect defense/pathogen-responsive transcripts, and, where possible, seed-specific transcripts for each transcript type including qPCR primers for transcripts encodingpolyphenol oxidase, 3 peroxidases, oxalate oxidase, 2 chitinases, NADPH oxidase (RbohA) and glutathione S-transferase. Defense gene expression assays have focused on wheat, because sequences for oat and wild oat spp. are underrepresented in public databases. (5) Goal 1B:Fungal transcriptome responses were evaluated forinteractions with aluminum toxicity and wild oat caryopsis colonization. (6)Goal 4: Real-time qPCR primers targeting the TEF-1α gene region and specific to the seed decay pathogen Fusarium avenaceum were developed, tested, and published. These primers also allowed for the detection and quantification of the Fusarium isolate F.a.1 in soils and seeds, thereby contributing to the soil-based assay for seed decay; these studies are completed with manuscripts in preparation.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Pollard, A.T. and Okubara, P.A. 2019. Real-time PCR quantification of Fusarium avenaceum in soil and seeds. Journal of Microbiological Methods 157, 21-30. https://doi.org/10.1016/j.mimet.2018.12.009
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Pollard, A.T. 2018. Seeds vs. enzymes: an enzymatic battle in the soil seedbank. Seed Science Research 28 (SI 3), 197-214. https://doi.org/10.1017/S0960258518000181
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Fuerst, E. Patrick, Matthew S. James, Anne T. Pollard, and Patricia A. Okubara, 2018. Defense enzyme responses in dormant wild oat and wheat caryopses challenged with a seed decay pathogen. Frontiers Plant Sci. 8. https://dx.doi.org/10.3389%2Ffpls.2017.02259
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Pollard, A.T. 2018. Harnessing the Beneficial Attributes of Soil Microorganisms for Sustainable Weed Management and Soil Fertility. Ph.D. dissertation, Washington State University, 287 pages.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Okubara P, Pollard A, Fuerst P (2018) Expression of defense enzymes and mRNAs in wild oat and wheat seeds challenged with the pathogen Fusarium avenaceum. 2018 Dryland Field Day Abstracts, Technical Report 18-1, p. 61-62.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ricky W. Lewis, Patricia Okubara, E. Patrick Fuerst, and Tarah S. Sullivan. Whole genome sequencing of Fusarium avenaceum strain F.a.1: a fungal strain showing biocontrol potential for wild oat. World Congress of Soil Science. Rio de Janeiro, Brazil, South America, August 14, 2018.
  • Type: Journal Articles Status: Other Year Published: 2019 Citation: Ricky W. Lewis, Patricia A. Okubara, E. Patrick Fuerst, Ruifeng He, David Gang, and Tarah S. Sullivan. Chronic Sublethal Aluminum Exposure and Avena fatua Caryopsis Colonization Influence Gene Expression of Fusarium avenaceum F.a.1. In Preparation for Molecular Plant-Microbe Interactions.
  • Type: Journal Articles Status: Other Year Published: 2019 Citation: Okubara PA, Fuerst EP. Expression of defense mRNAs in wheat seeds challenged with the pathogen Fusarium avenaceum. Manuscript in Preparation for Frontiers in Plant Science.
  • Type: Journal Articles Status: Other Year Published: 2019 Citation: Pollard, A.T., Okubara, P.A., and Fuerst, E.P. Interaction between dormant wild oat and Fusarium avenaceum in soil- Part I. Caryopses. Plant and Soil. To be submitted Spring 2019.
  • Type: Journal Articles Status: Other Year Published: 2019 Citation: Pollard, A.T., Okubara, P.A., and Fuerst, E.P. Interaction between dormant wild oat and Fusarium avenaceum in soil- Part II. Whole seeds. Plant and Soil. To be submitted Spring 2019.


Progress 01/01/17 to 12/31/17

Outputs
Target Audience:Research communities in seed longevity and survival; weed management research communities; native plant seed producers.? Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One-on-one training of our PhD student, Ms. Pollard. Dr. Fuerst participated in the Annual Meeting of the Weed Science Society of America, Tucson, AZ, February 6-9, 2017. Dr. Okubara participated in meetings: Plant Animal Genome XXIV, January 2017, San Diego, CA; International Plant Science Conference, September 2017, Kiel, Germany. Ms. Pollard attended 12th Triennial Conference of the International Society for Seed Science, Monterey, CA, September 10-14, 2017, and the BIOAg Symposium, Pullman, WA, March 1, 2017. Dr. Sullivan attended the annual meeting of the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. Dr. Okubara and Ms. Pollard supervised a female intern (an entering undergraduate) in summer 2017 in the USDA-WSU Upward Bound Bridge program 'Pumping-Up the Math and Science Pipeline' in Pullman, WA. The research entailed aspects of the development of soil-based assays for seed decay (Goal 4), and included extracting soil DNA, preparing solutions, setting-up and analyzing seed viability experiments, making media, and performing serial dilution plating. We guided the mentee in performing laboratory techniques, and oversaw the professional development of the mentee through co-attendance at departmental seminars and coaching mentee's public speaking and presentation skills. Drs. Sullivan and Lewis also guided an additional female summer intern in the same program. The research was focused on examining enzyme responses of wheat and wild oat seeds to novel, currently unidentified soil fungi. The intern was trained in sterile technique, culturing fungi, and performing enzyme assays using a microplate reader. Additionally, the mentee gained training in public speaking and presentation skills. How have the results been disseminated to communities of interest? Dr. Fuerst gave an oral presentation on results at annual meeting of the Weed Science Society of America, Tucson, AZ. (See abstract citation under "Products".) Ms. Pollard gave three presentations, two at the International Society for Seed Science (an oral presentation and a poster), Monterey, CA, and the BIOAg Symposium, Pullman, WA (poster). (See abstract citations under "Products".) What do you plan to do during the next reporting period to accomplish the goals?Goal 1B: Complete experiments on the effect of F.a.1 on defense gene expression in dormant wheat seeds using new real-time PCR assays. Provisional manuscript titles: Okubara PA, Pollard AT, Fuerst EP (2018) Real-time polymerase chain reaction quantification of defense-related transcripts in wheat seeds. Okubara PA, Pollard AT, Fuerst EP (2018) Action of Fusarium avenaceum on defense gene expression in dormant wheat seeds. Goal 1: Leachates generated from wild oat seeds exposed to F.a.1 cultured in the presence of aluminum will be examined for chitinase and polyphenol oxidase activity. Leachates and/or caryopses will then be examined via metabolomic profiling to determine the underlying metabolic mechanisms of pathogenicity and the effects of aluminum on the metabolome. To elucidate the role of diffusible factors in F.a.1 pathogenicity, studies have been initiated where polypropylene membranes will be used to physically, but not chemically, separate caryopses from contact with F.a.1. Leachates from caryopses will then be evaluated for enzyme activity. Goal 4: Prepare and submit manuscript: Provisional manuscript title: Pollard, A.T. and Okubara, P.A. (2018) Real-time PCR for the detection and quantification of Fusarium avenaceum in soils and seeds. Journal of Microbiological Methods. To be submitted February 2018.

Impacts
What was accomplished under these goals? (1) E.P. Fuerst and P. Okubara supervised Soil Science PhD student Anne Pollard in 2017. Anne has made great strides in her research, nearly concluding two research sections of her dissertation. The introduction was completed and submitted to Seed Science Research as a refereed publication. (2) T Sullivan supervised Crop & Soil Science postdoctoral research associate, Rick Lewis, July 1 - December 31, 2017. (3) Goal 1A: We concluded extensive assays assessing biochemical defenses of seeds including polyphenol oxidase, peroxidase, chitinase, and oxalate oxidase in wheat and wild oat; results are being published in a refereed journal, in press. (4) Goal 1B: Additional primers were designed to detect defense/pathogen-responsive transcripts, and, where possible, seed-specific transcripts for each transcript type including reverse transcription qPCR primers for transcripts of peroxidase, oxalate oxidase, chitinase, NADPH oxidase (RbohA) and glutathione S-transferase. Defense gene expression assays have focused on wheat, because sequences for oat and wild oat spp. are underrepresented in public databases. (5) Goal 4: Real-time qPCR primers targeting the TEF-1α gene region and specific to the seed decay pathogen Fusarium avenaceum have been developed and tested. These primers will allow for the detection and quantification of the Fusarium isolate F.a.1 in soils and seeds, thereby enhancing the efficacy of the developed soil-based assay for seed decay (Goal 4). A manuscript detailing this primer development work is currently in progress and targeted for journal submission by February 1, 2018. From several previous soil-based experiments, total genomic DNA has been extracted from pasteurized and non-pasteurized soil and the wild oat seeds (caryopses and hulls) incubated therein. Currently, real-time qPCR using our novel F. avenaceum primers is being conducted on these DNA samples to assess fungal concentrations throughout the duration of the 4-9 wk soil-based assays. Additional experiments have been conducted on 1) wild oat seed viability following an incubation time course in pasteurized or non-pasteurized soil ± F.a.1; 2) assessment of emissions of volatile organic compounds from F.a.1; and 3) replicating previous soil-based experiments on the defense enzyme activity of wild oat caryopses and whole seeds in response to F.a.1. Work this spring will focus on analyzing soil enzyme activity in the presence of the seed decay pathogen F.a.1 and dormant wild oat seeds in soils with diverse physical and chemical properties. (6) Dr. Lewis has sequenced the F.a.1 genome in-house using Next Generation Sequencing technology (Illumina MiSeq). Assembly of the genome along with annotation is ongoing. Sequences were obtained for several other fungi isolated from soil and genome assembly is in progress as well. Genome assemblies will be made available via the NCBI database and the genome assembly of F.a.1 will be published along with data gathered from the experiments outlined below. Investigation was initiated concerning the effects of aluminum toxicity on the interaction between F.a.1 and wild oat caryopses. This is particularly relevant to agriculture in eastern Washington, where in some regions soils are becoming increasingly acidified, leading to aluminum toxicity. Methods have been developed regarding aluminum exposure of F.a.1 and the appropriate aluminum concentrations are now being determined.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Fuerst, E. Patrick, Matthew S. James, Anne T. Pollard, and Patricia A. Okubara, 2018. Defense enzyme responses in dormant wild oat and wheat caryopses challenged with a seed decay pathogen. Frontiers Plant Sci. (In Press, accepted Dec 27, 2017).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: E. Patrick Fuerst, Matthew S. James, Anne T. Pollard, Patricia A. Okubara, G.N.M. Kumar, and Craig F. Morris. Induction of Biochemical and Molecular Seed Defenses by a Seed Decay Pathogen in Dormant Wild Oat and Wheat Caryopses. Oral presentation at the Annual Meeting of the Weed Science Society of America, Tucson, AZ, February 6-9, 2017. Available at http://wssaabstracts.com/public/45/proceedings.html (accessed January 9, 2018).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Pollard, A.T., Okubara, P., James, M., and Fuerst, E. P. 2017. Biochemical and molecular regulation of defense responses to a pathogenic soil fungus in dormant seeds. Oral presentation at the 12th Triennial Conference of the International Society for Seed Science, Monterey, CA, September 10-14, 2017. [Online]. Available at isss2017.ucdavis.edu/wp-content/uploads/2017/09/ISSS_Full_Abstracts_online.pdf (Accessed 6, December 2017).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Pollard, A.T, Fuerst, E. P., and Okubara, P. 2017. Biochemical and molecular regulation of defense responses to a pathogenic soil fungus in dormant weed seeds. Poster presentation at the 12th Triennial Conference of the International Society for Seed Science, Monterey, CA, September 10-14, 2017. Available at http://isss2017.ucdavis.edu/wp-content/uploads/2017/09/ISSS_Full_Abstracts_online.pdf (Accessed 29, December 2017).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Pollard, A.T., Fuerst, E.P., and Okubara, P. 2017. Fusarium avenaceum: A potential tool to combat wild oats in the soil weed seedbank. Poster presentation at the BIOAg Symposium, Pullman, WA, March 1, 2017. Available at http://csanr.wsu.edu/wp-content/uploads/2017/03/Pollard.pdf (Accessed 29, December 2017).


Progress 01/01/16 to 12/31/16

Outputs
Target Audience: Research communities in seed longevity and survival; weed management research communities; native plant seed producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One-on-one training of both of our PhD students. Dr. Okubara attended meetings: Plant Animal Genome XXIII, Jan 2016, San Diego, CA; American Phytopathological Society Pacific Division Annual Meeting, Mt Vernon, WA; and the International Society for Molecular Plant-Microbe Interactions Congress, Portland, OR Dr. Sullivan and Ms. Pollard attended the annual meeting of the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Phoenix, AZ. Dr. Okubara and Ms. Pollard supervised a female summer intern (an entering undergraduate) in the USDA-WSU Upward Bound Bridge program 'Pumping-Up the Math and Science Pipeline' in Pullman, WA. The research entailed aspects of the development of soil-based assays for seed decay (Objective 4) and sequencing of the F.a.1 ITS DNA for primer design. We guided the mentee in performing laboratory and greenhouse techniques, and oversaw the professional development of the mentee through co-attendance at departmental seminars and coaching mentee's public speaking and presentation skills. How have the results been disseminated to communities of interest?Ms. Pollard presented results at annual meeting of the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America (abstract cited in "Products"). • Mr. James presented results at Tilth Producers annual conference (abstract cited in "Products"). What do you plan to do during the next reporting period to accomplish the goals? Objective 1A: Conclude research and submit manuscript on induction of defense enzyme activities during pathogen challenge of wild oat and wheat and present results at local and national meetings. Dr. Fuerst will present results at the 2017 annual meeting of the Weed Science Society of America. Objective 1B: Complete experiments for a manuscript on seed defense gene expression using real-time PCR and present results at a national meeting. Objective 4: Conclude soil-based assay study for seed decay and seed defense responses and present results at a national meeting. Additionally, Ms. Pollard will be presenting this work at two local meetings, the BIOAg Symposium and the Graduate and Professional Students Association Research Exposition, both at (WSU, Pullman, WA) in March 2017.

Impacts
What was accomplished under these goals? (1) E.P. Fuerst and P. Okubara supervised Soil Science PhD student Anne Pollard in 2016. Anne has completed coursework and is making great strides in research. (2) E.P. Fuerst supervised Crop & Soil Science PhD student Matthew James in 2016. Matthew made great progress in his research. (3) Objective 1A: We conducted extensive assays assessing biochemical defenses of seeds including polyphenol oxidase, peroxidase, chitinase, and oxalate oxidase in wheat and wild oat. (4) Objective 1B: Continued to design and test real-time PCR primers for mRNAs encoding polyphenol oxidase, peroxidase, oxalate oxidase, chitinase, NADPH oxidase, EFTu-1alpha and glyceraldehyde 3-phosphate dehydrogenase. Reverse transcription qPCR indicated that F.a.1 induced polyphenol oxidase and peroxidase mRNA without detectable induction in the no-pathogen control, and a 4.2-fold reduction of oxalate oxidase mRNA relative to the internal standard glyceraldehyde 3-phosphate dehydrogenase in wheat caryopses. Polyphenol oxidase, but not peroxidase, was induced in wild oat caryopses. In a separate experiment, NADPH oxidase mRNA was induced 4.2-fold in wheat seeds, whereas chitinase mRNA was induced 6.8-fold in wild oat. Our findings indicate differential responses of wild oat and wheat to F.a.1. (5) Objective 4: Developed soil-based assay for seed decay and seed defense responses utilizing the new biochemical tools and protocols we developed, above. Induction of defense enzymes polyphenol oxidase, peroxidase, oxalate oxidase, and exochitinase in wild oat whole caryopses was assessed following incubation in field soil ± F.a.1 at 15 °C or 25 °C. Peak defense enzyme induction occurs more quickly at 25 °C than at 15 °C. F.a.1 inoculated soil causes significant seed decay occur, with decay onset and extent more pronounced at 25°C. In additional experiments, induction of polyphenol oxidase, peroxidase, and exochitinase was evaluated in separate hull and caryopsis seed fractions following incubation at 15 °C on either F.a.1 cultures on PDA or in F.a.1-inoculated soil. Significantly greater exochitinase induction occurs in the hull compared to the caryopsis. Experiments and analysis are ongoing. The ITS and TEF-1a gene regions of F.a.1 were sequenced and species-specific F.a.1 primers are in development.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: James, Matthew, E. Patrick Fuerst, Patricia Okubara, and Craig Morris. 2016. Induction of defense enzymes in dormant wild oat (Avena fatua) and wheat (Triticum spp.) by a seed-pathogenic fungus. Poster presented at Tilth Producers annual conference, November 11-13, Wenatchee, WA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Pollard, Anne, E. Patrick Fuerst, and Patricia Okubara. 2016. Fusarium avenaceum: A Potential Tool to Combat Wild Oats in the Soil Weed Seedbank. Poster presented at the annual meeting of the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. Available at https://scisoc.confex.com/crops/2016am/webprogram/Paper102514.html.


Progress 01/01/15 to 12/31/15

Outputs
Target Audience:Research communities in seed longevity and survival; weed management research communities; native plant seed producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One-on-one training of both of our PhD students. Dr. Okubara attended two meetings: Plant Animal Genome XXIII, Jan 10-14, 2015, San Diego, CA and 9th Symposium of the International Society for Root Research, Oct 6-9, 2015, Canberra, Australia. Dr. Sullivan attended the annual meeting of the American Society of Agronomy, Crop Science Society of America, and Soil Science Society How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Objective 1A: Conclude research and submit manuscript on induction of enzyme activities during pathogen challenge of wild oat and wheat seeds and present results at a national meeting. Objective 1B: Complete experiments for a manuscript on seed defense gene expression using real-time PCR. Objective 4: Develop soil-based assay for seed decay and seed defense responses utilizing

Impacts
What was accomplished under these goals? (1) Supervised Soil Science PhD student Anne Pollard in 2014 and 2015; Tarah Sullivan is major advisor. Anne has made great strides in coursework and a great start in research. (2) Recruited Crop & Soil Science PhD student Matthew James in 2015; E. Patrick Fuerst is major advisor. Matthew has had a successful first semester coursework and has made great progress in his research. (3) Objective 1A: We developed the methods needed to assess biochemical defenses of seeds by developing high throughput assays for polyphenol oxidase, peroxidase, chitinase, and oxalate oxidase. (4) Objective 1B: Designed and tested real-time PCR primers for transcripts encoding polyphenol oxidase, peroxidase, oxalate oxidase, chitinase, NADPH oxidase, and a housekeeping transcript (for use as an internal standard). Developed mRNA extraction method from seeds incubated plus and minus pathogen. Ran initial RT-PCR studies indicating induction of mRNA for polyphenol oxidase and NADPH oxidase.

Publications


    Progress 01/01/14 to 12/31/14

    Outputs
    Target Audience: Research communities in seed longevity and survival; weed management research communities; native plant seed producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Anne Pollard, PhD student, started working on the seed decay project under Tarah Sullivan in December, 2014. We are recruiting one more graduate student. E.P. Fuerst obtainedprofessional training while attending annual meetings of the Weed Science Society of America. How have the results been disseminated to communities of interest? A refereed publication and a report at national weed science meeting. See citations under "Products," above. What do you plan to do during the next reporting period to accomplish the goals? Recruit one more graduate student for the 'Seed Decay' project. Utilize the new biochemical and molecular tools and protocols we have developed to assess seed defenses as part of this project.

    Impacts
    What was accomplished under these goals? (1) Secured USDA/NIFA proposal was funded, 2013-01855 starting January 1, 2014. Title: 'Biochemical And Molecular Mechanisms Of Resistance To Decay In Weed Seeds', E.P. Fuerst (PD), P.A. Okubara, and T. Sullivan. (2) Published invited refereed journal article. See "Products", above. (3) Recruited PhD student Anne Pollard, under Tarah Sullivan. We continue efforts to recruit another graduate student. (4) Advanced the innovative methods that are be used to assess biochemical and molecular defenses of seeds.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2014 Citation: E. Patrick Fuerst, Patricia A. Okubara, James V. Anderson, and Craig F. Morris. 2014. Polyphenol oxidase as a biochemical seed defense mechanism. Frontiers in Plant Science (Invited manuscript) (Hypothesis and Theory Article). doi: 10.3389/fpls.2014.00689.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: E. Patrick Fuerst, Ann C. Kennedy, Patricia A. Okubara, James V. Anderson, and Robert S. Gallagher. 2014. Activation of Biochemical Defenses by a Seed Decay Pathogen in Dormant Wild Oat Seeds and Caryopses. (Presentation and abstract.) Proceedings. Weed Science Society of America, Vancouver, BC, February 3-5, 2014. (This was also the Project Director meeting for the NIFA AFRI weed seed decay project.) NIFA support was acknowledged